CIENCIA 7(1), 72-77, 1999 Maracaibo, Venezuela Phytochemical evaluation and antibacterial activity of () inflorescences Carmen de los Ríos1*, Doris Hidalgo Báez1, Quilianio Contreras1, Oscar Crescente2 and Adela Caserta2 1Departamento de Química, Facultad de Ciencias, Universidad de Los Andes. Mérida, Venezuela. 2Escuela de Ciencias, Departamento de Química, Universidad de Oriente. Cumaná, Venezuela

Recibido: 22-06-98 Aceptado: 26-03-99

Abstract The increasing interest for the medicinal properties in is what induced the present work and was selected for the ethnobotanical information the species Espeletia schultzii Wedd. (Frailejón de Octubre), of the family Asteraceae, to accomplish an in vitro evaluation of its anti- biotic and phototoxic activities. This species is used for the treatment of asthma in folk medi- cine in the Venezuelan Andes. A phytochemical evaluation was carried out which confirmed that the inflorescences extract contains kaurenic acids, it was isolated and identified benzo- phenone and two flavonoids, identified as quercetin and its 3-0-galactoside. These are the first flavonoids found in this species, and this is the first report of benzophenone in Espeletia. Three fractions obtained of the ethyl acetate extract, as well as the aqueous residual extract, were selected to accomplish an in vitro evaluation of their antibiotic and phototoxic activities. These activities were tested employing bacteria of clinical origin. Only one of the ethyl acetate fractions and the aqueous residual were found active against at least one bacterial strain. As far as we know, this study constitutes the first report on evaluation of the antibacterial activity of E. schultzii Wedd. Key words: Antibacterial activity, Asteraceae, Espeletia shultzii, flavonoids, treatment asthma. Evaluación fitoquímica y actividad antibacteriana de las inflorescencias de Espeletia schultzii (Asteraceae)

Resumen El presente trabajo es el producto del actual y creciente interés por las plantas medicina- les, y basándose en la exploración etnobotánica se escogió la especie Espeletia schultzii Wedd. (Frailejón de Octubre), Asteraceae, para evaluar in vitro su actividad antibacteriana y fototóxi- ca. Esta especie es utilizada entre la población andina como un remedio para el asma. La eva- luación fitoquímica realizada a las inflorescencias confirmó la presencia de ácidos kaurénicos y se reporta el aislamiento e identificación de ácidos kaurénicos, benzofenona y dos flavonoides, identificados como la quercetina y su 3-O-galactósido. Estos son los primeros flavonoides aisla- dos en esta especie, y éste es el primer reporte de benzofenona en el género Espeletia. Tres frac- ciones obtenidas del extracto de acetato de etilo, así como, el extracto acuoso residual, fueron

* To whom correspondence should be addressed.

Scientific Journal from the Experimental Faculty of Sciences, Volume 7 Nº 1, January-April 1999 C. de los Ríos et al. / Ciencia Vol. 7, Nº 1 (1999) 72-77 73 seleccionados para evaluar sus actividades antibacteriana y fototóxica. Estas actividades se ensayaron con varias bacterias de origen clínico. Sólo una fracción del extracto de acetato de etilo y el extracto acuoso residual mostraron actividad antibacteriana contra alguna cepa bac- teriana. Este trabajo constituye el primer reporte de estudios realizados sobre la actividad anti- bacteriana de la especie Espeletia schultzii Wedd. Palabras clave: Actividad antibacteriana; asma; Asteraceae; Espeletia shultzii; flavonoides.

Introduction with organic solvents of increasing polarity and water. The extracts were analyzed by bi- On the basis of ethnobotanical infor- dimensional paper chromatography with Espeletia schultzii mation, the species TBA (terbuthanol: acetic acid: water 3:1:1) Wedd. (Frailejón de Octubre), of the family and acetic acid (15 %) as eluents. The chro- Asteraceae, was selected for an in vitro matograms were visualized with NH 3. The evaluation of its antibiotic and phototoxic dichloromethane extract (40.4 g) was frac- activities. This species is widely distributed tionated in a silica gel column, 28 fractions in the Páramo, Mérida State, Venezuela, were collected and similar fractions were about 2.900-4.300 m (1), and is used as a combined from which three compounds remedy for the treatment of asthma in the were isolated. The ethyl acetate extract (50.3 folk medicine of the Venezuelan Andes. g) was submitted to various sephadex LH-20 Chemical studies carried out on this and polyamide column chromatography, reported the isolation and identification of thirty-four fractions were collected and kaurenic acids, hydroxyacetophenone de - similar fractions were combined from which rivatives (2, 3, 4), and qualitative detection two compounds were isolated and purified of glycosylated flavonoids (5). In the present using sephadex LH-20 columns. study the inflorescences of this plant were evaluated phytochemically and biologically. Spectroscopic data Material and Methods Compound 1. Benzophenone: -1 White powder. IR max cm : 3088, Plant collection 1653, 1448, 1416, 919, 814, 775. 1HNMR (200 MHz): 7.76 (2H, dd, H-3), 7.64 (1H, tt, The Espeletia schultzii Wedd. (- 13 aceae) species was collected in Venezuela, H-5), 7.52 (2H, td, H-4). CNMR (50 MHz): Mérida State, Municipio Justo Briceño, 198.60 (C-1), 138.95 (C-2), 130.88 (C-3), Páramo of Piedras Blancas, about 13 Km 129.45 (C-4), 133.70 (C-5). from the Pico El Aguila, toward Piñango way Compound 2. Grandifloric acid (Contreras 157). The specimen was taxo- ent-15- -hydroxy-16-en-kaur-19-oic nomically classified by Forest technician Gi- acid: useppe Adamo, Centro Jardín Botánico, -1 Facultad de Ciencias, Universidad de Los White powder. IR max cm : 3500, + Andes, and a voucher specimen, # 2527, is 3303, 2983, 1692. MS m/e: 318 (M ). 1 kept in the MERC Herbarium of this Fac- HNMR (200 MHz, C5 D5 N): 5.52 and 5.23 ulty. (1H, s, H-17), 4.18 (1H, s, H-15), 2.75 (1H, m, H-13), 1.20 (3H, s, H-18), 1.37 (3H, s, 13 Extraction and isolation H-20). CNMR (50 MHz): 41.2 (C-1), 19.9 About 900 g of the inflorescences dried (C-2), 38.8 (C-3), 44.0 (C-4), 57.3 (C-5), 22.1 and ground were submitted to extraction (C-6), 36.7 (C-7), 48.4 (C-8), 54.1 (C-9), 40.3 (C-10), 18.8 (C-11), 33.1 (C-12), 42.9 (C-13),

Scientific Journal from the Experimental Faculty of Sciences, Volume 7 Nº 1, January-April 1999 74 Phytochemical evaluation and antibacterial activity of Espeletia schultzii inflorescences

36.4 (C-14), 82.7 (C-15), 161.0 (C-16), 107.7 (C-6´), 120.97 (C-1´), 115.82 (C-5´), 115.07 (C-17), 29.3 (C-18), 180.1 (C-19), 16.3 (C-2´), 103.8 (C-10), 98.5 (C-6), 93.4 (C-8), (C-20). 101.7 (C-1´´), 75.7 (C-5´´), 73.07 (C-3´´), 71.09 (C-2´´), 67.8 (C-4´´), 60.0 (C-6´´). Compound 3. Grandiflorenic acid [ent-kaur-9 (11),16-dien-19-oic acid]: -1 White powder. IR max cm : 3374, 3100, 2976, 1692, 950-876. 1HNMR (200 MHz): 1.02 and 1.24 (3H, s, H-18 and H-20), 2.77 (1H, m, H-13), 5.24 (1H, t br, H-11), 4.91 and 4.79 (1H, s, H-17). 13CNMR 1 (50 MHz): 40.85 (C-1), 20.19 (C-2), 38.32 (C-3), 44.79 (C-4), 46.70 (C-5), 18.52 (C-6), 29.76 (C-7), 42.31 (C-8), 156.02 (C-9), 38.85 (C-10), 114.05 (C-11), 37.95 (C-12), 41.30 (C-13), 45.03 (C-14), 50.39 (C-15), 158.50 (C-16), 105.44 (C-17), 28.25 (C-18), 184.10 (C-19), 23.59 (C-20). Compound 4. Quercetin:

Yellow powder. UV max nm: (MeOH) 2 372, 300 sh, 257; (NaOMe) 412, 328, 260; (AlCl3) 443, 273; (AlCl3/HCl) 430, 355 sh 308 sh, 270; (NaOAc) 400, 328, 275; 1 (NaOAc/H3 BO3) 390, 293, 260. HNMR (200 MHz, DMSO-d6): 12.5 ( 1H, s, OH in C-5), 10.8 (1H, s, OH in C-3), 9.6 (1H, s, OH in C-7), 9.3 (1H, s, OH in C-3´/C-4´), 7.7 (1H, d, J= 2.2 Hz, H-2´), 7.5 (1H, d d, J= 2.2, 9 Hz, H-6´), 6.9 ( 1H, d, J= 9, H-5´), 6.4 ( 1H, d, 3 J=2.2, H-8), 6.2 (1H, d, J= 2.3, H-6). 13 CNMR (50 MHz, DMSO-d6): 146.68 (C-2), 135.60 (C-3), 175.71 (C-4), 160.69 (C-5), 98.05 (C-6), 163.75 (C-7), 93.23 (C-8), 156.01 (C-9), 102.89 (C-10), 121.83 (C-1´), 114.93 (C-2´), 144.93 (C-3´), 147.57 (C-4´), 115.47 (C-5´), 119.85 (C-6´). Compound 5. Quercetin 3-O- galactoside: 4

UV max nm: (MeOH) 358, 298 sh, 270 sh, 257; (NaOMe) 405, 325 sh, 272; (AlCl3) 434, 335 sh, 300, 272; (AlCl3/HCl) 402, 360 sh, 303 sh, 272; (NaOAc) 390, 320 sh, 270; 13 (NaOAc/H3 BO3) 373, 295sh, 260. CNMR (50 MHz, DMSO-d6): 177.4 (C-4), 164.03 (C-7), 161.1 (C-5), 156.2 (C-2, C-9), 148.3 5 (C-4´), 144.7 (C-3´), 133.4 (C-3), 121.86

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Antibacterial Activity kaur-9 (11),16-dien-19-oic acid] (3) (100.3 mg). The structures of the compounds were Three fractions obtained of the ethyl established by the spectral methods: UV, 1H acetate extract, as well as the aqueous re- NMR, 13C NMR, IR, and TLC was compared sidual extract, were selected for an in vitro with an authentic sample. All spectral data evaluation of the antibacterial activity using were compared with those reported in the the disc-diffuse method (6), and the photo- literature (8, 9). Two flavonols were obtained toxicity was performed by the Meckes from the ethyl acetate extract. The analysis method (7). These activities were tested em- of the UV spectra in MeOH and displace- ploying bacteria of clinical origin, Gram (+) ment reactants, carried out on the isolated and Gram (-). flavonols, indicated that these had the same Paper discs impregnated with 25 µl of pattern of substitution in the positions 5, 7, the extract (corresponding to 40 mg/mL), as 3’ and 4’, and that the substitution in posi- tion 3 was different. Comparison of their well as discs impregnated with the solvents 1 13 used to prepare each solution were placed spectroscopic data UV, H NMR, C NMR, on sterile Müller-Hinton agar plates, which IR and MS with those reported in the litera- were inoculated with test bacteria, Bacillus ture (10, 11) and TLC with an authentic cereus, Staphylococcus aureus, Salmonella sample allowed the identification as quer- typhi, Escherichia coli and Pseudomonas ae- cetin (4) (25.2 mg) and its 3-0-galactoside (5) ruginosa. The plates were pre-incubated for (18.3 mg). 18 h at 4°C to allow diffusion, and then were One of the fractions of the ethyl acetate kept 24 h in the incubator at 37°C. The sizes extract and the aqueous residual extract of the inhibition zones were measured. To showed antibacterial activity against Bacil- test for light-activated antibacterial activity, lus cereus and Staphylococcus aureus, re- one replicate was exposed to UV-A light (200 spectively (Tables 1 and 2). W, = 320-400 nm, from Hanovia lamps) for 2, 4, 6, 8 h, before they were placed on the The exposure to UV-A light had no ef- nutrient agar inoculated with test bacteria, fect on the activities of the extracts. then submitted to the procedure mentioned before. All assays were performed in dupli- Discussion cate. Espeletia schultzii Wedd. is used for the treatment of asthma, which suggested that Results some biological activity could be present in The bidimensional paper chromatogra- this plant. The present experimental study phy of the organic extracts obtained from revealed that some extract obtained of this the inflorescences showed that flavonoids of plant showed antibacterial activity against different polarities were present. The dichlo- B. cereus and S. aureus in vitro. This result romethane and ethyl acetate extract were could be attributed to some components fractionated with organic solvents and water with biological activity present in this plant. followed by chromatographic separation The flavonoids have antibacterial properties and the chemical structures of the com- and it could be that the observed antibacte- pounds obtained were determined. Three rial activity is related to the presence of fla- compounds were isolated from the dichloro- vonoids in the plant. The antibacterial activ- methane extract and identified as benzo- ity of the 3-O-galactoside has been reported phenone (1) (40.2 mg), grandifloric acid in the literature (12). Several investigations ent-15-b-hydroxy-16-en-kaur-19-oic acid carried out on Quercetin report its antiviral (2) (80.5 mg), and grandiflorenic acid [ent- properties (13). The genus Espeletia is char- acterizated by the ocurrence of kaurenic ac-

Scientific Journal from the Experimental Faculty of Sciences, Volume 7 Nº 1, January-April 1999 76 Phytochemical evaluation and antibacterial activity of Espeletia schultzii inflorescences

Table 1 Antibacterial and phototoxic activities of one of the ethyl acetate fractions of the inflorescences from Espeletia schultzii Wedd

Zone of Inhibition (mm) Microorganism Irradiation Time (h) 0 h 2 h 4 h 6 h 8 h E. coli ————— P. aeruginosa ————— S. typhi ————— S. aureus ————— B. cereus 14 12 14 12 14 — No Zone of Inhibition was observed. Table 2 Antibacterial and phototoxic activities of the aqueous extract of the inflorescences from Espeletia schultzii Wedd

Zone of Inhibition (mm) Microorganism Irradiation Time (h) 0 h 2 h 4 h 6 h 8 h E. coli ————— P. aeruginosa ————— S. typhi ————— S. aureus 15 14 12 — — B. cereus ————— — No Zone of Inhibition was observed. ids. Grandiflorenic acid [ent-kaur-9 (11)-16- this species. This is the first report of benzo- -en-19-oic acid], which is common in species phenone in genus Espeletia. of Espeletia (14), and has been previously As far as we know, this study consti- isolated from E. schultzii (2, 3, 4), is also pres- tutes the first report on evaluation of the an- ent in Montana tomentosa “Zoapatle” a plant tibacterial and phototoxic activities of Espe- used in Mexican folk medicine, which is re- letia schultzii Wedd. ported as an anticonceptive (15). The evidence of antibacterial activity Acknowledgements and the presence of compounds with known biological activity in the studied plant indi- We thank the Consejo de Desarrollo cate that E. schultzii could be a therapeutic Científico, Humanístico y Tecnológico, source in the treatment of some infectious CDCHT, of the Universidad de Los Andes, diseases. Mérida, Venezuela, for financial support of this research as well the Consejo de Investi- The flavonoids quercetin and its 3-0- gación de la Universidad de Oriente, Cuma- galactoside are the first flavonoids found in ná, Venezuela, and CONICIT (S1-2202).

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Thanks are due to Betty Ramírez and Ale- 9. REYNOLDS W., ENRÍQUEZ R., ESCOBAR jandro Fleitas, of the Universidad de Los An- L., LOZOYA X. Can J Chem 62: 2421-2425, des, for typing and editing this manuscript. 1984. 10. MARKHAM K.R., MOHAN CHARI V. in The References Flavonoids Advances in Research, Har- borne J.B., Mabry T.J. (Eds.), Chapman 1. VARESCHI V. Flora de los Páramos de and Hall, London (England), 1:19-134, Venezuela. Ediciones Universidad de Los 1982. Andes, Mérida (Venezuela), pp 298, 1970. 11. MARKHAM K.R. in Methods in Plant Bio- 2. KLOSS P. Arch der Pharm 376-381, 1968. chemistry, Dey P.M., Harborne J.B. (Eds.), 3. BRIESKORN C., PÖHLMANN E. Chem Ber Academic Press, 1:197-235, 1991. 102: 2621-2628, 1969. 12. WAAGE S., HEDIN P. Phytochemistry 24 4. BOHLMANN F., RAO N. Chem Ber 106 (9): (2): 243-245, 1985. 3035-3038, 1973. 13. VLIETINCK A., VANDEN BERGHE D., 5. MORENO M., HURTADO J., SILVA J., HAEMERS A. in Plant Flavonoids in Biol- FARIÑAS M., AZÓCAR A. Ecotrópicos 3 (2): ogy and Medicine II: Biochemical, Cellu- 56-5, 1990. lar and Medicinal Properties, Cody V. et 6. BAUER A., KIRBY W., SHERRIS J., TURK al., (Eds.), Alan R. Liss Inc., New York M. Am J Clin Path 45 (4): 493-496, 1966. (USA), pp. 283-299, 1988. 7. MECKES M., VILLARREAL M. L., TORTO- 14. LEWIS N., MACMILLAN J. J Chem Soc RIELLO J. Phytother Res 9: 244-250, Perkin 1:1270-1278 1980. 1995. 15. LOZOYA X., ENRIQUEZ R., BEJAR E., ES- 8. HUTCHINSON M., LEWER P., MACMIL- TRADA A., GIRÓN H., PONCE-MONTER H., LAN, J. J Chem Soc Perkin 2363-2366, GALLEGOS A. Contraception 27 3: 267- 1984. 279, 1983.

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